It is known that droplets with a volume of more than 10 μl can be dispensed from the air very easily, since if the pipette is correctly manipulated, the droplets leave the pipette tip of their own accord. The droplet size is then determined by the physical properties of the sample liquid, such as surface tension or viscosity. The droplet size thus limits the resolution of the quantity of liquid to be dispensed.
The aspirating and dispensing, i.e. the pipetting of liquid samples with a volume of less than 10 μl, in contrast, typically requires instruments and techniques which guarantee the dispensing of such small samples. The dispensing of a liquid with a pipette tip, i.e. with the endpiece of a device for aspirating and/or dispensing sample liquid, can occur from the air (“from air”) or by touching a surface. This surface can be the solid surface of a container (“on tip touch”), into which the liquid sample is to be dispensed. It can also be the surface of a liquid in this container (“on liquid surface”). A mixing procedure following the dispensing is recommended—particularly for very small sample volumes in the nanoliter or even picoliter range—so that uniform distribution of the sample volume in a diluent is ensured.
Systems for separating samples from a liquid are known as pipettors. Such systems serve, for example, for dispensing liquids into the wells of Standard Microtitration Plates™ (trademark of Beckman Coulter, Inc., 4300 N. Harbour Blvd., P.O. Box 3100 Fullerton, Cailf., USA 92834) and/or microplates with 96 wells. The reduction of the sample volumes (e.g. for filling high-density microplates with 384, 864, 1536, or even more wells) plays an increasingly important role, with the precision of the sample volume dispensed being of great importance. The elevation of the number of samples typically also requires miniaturization of the experiment, so that the use of a pipettor is necessary and special requirements must be placed on the precision of sample volumes and the accuracy of the movement control and/or of the dispensing of this pipettor.
Disposable tips significantly reduce the danger of unintentional transfer of parts of the sample (contamination). Simple disposable tips are known (so-called “air-displacement tips”), whose geometry and material is optimized for the exact aspirating and/or dispensing of very small volumes. The use of so-called “positive-displacement tips”, which have a pump plunger inside, is also known.
For automation, two procedures must be differentiated from one another: the defined aspiration and the subsequent dispensing of liquid samples. Between these procedures, typically the pipette tip is moved by the experimenter or by a robot, so that the aspiration location of a liquid sample is different from its dispensing location. For the precision of dispensing and/or aspiration/dispensing, only the liquid system is essential, which comprises a pump (e.g. a diluter implemented as a syringe pump), tubing, and an endpiece (pipette tip).
The precision (ACC=accuracy) and reproducibility (CV=coefficient of variation) of the dispensing and/or aspiration/dispensing of a liquid sample can be influenced by greatly varying parameters. The speed of dispensing largely determines, for example, how the droplet breaks away from the pipette tip.
In principle, two basic modes are differentiated in pipetting: single pipetting and multipipetting. In the single pipetting mode, a liquid sample is aspirated and dispensed at another location. In the multipipetting mode, a larger volume of liquid is aspirated at one time and subsequently dispensed in several—typically equivalent—portions (aliquots) at one or more different locations, e.g. in various wells of a Standard Microtitration Plate™.
The measurement of the volume of a liquid sample, however, does not take into consideration the way in which a droplet was separated: in Europe, the norm ISO/DIS 8655-1 of the International Organization for Standardization (ISO) (whose main offices are in Geneva, Switzerland) has been available at least in draft form since 1990. This norm defines the basic conditions for performing laboratory work with dispensing devices, such as pipettes, dispensers, and burettes. Known national norms, such as ASTM (USA), British Standard (GB), or the newest draft DIN 12650 (Germany), have to fit themselves into the system of the ISO norm ISO/DIS 8655-1.
The norm DIN 12650 essentially differentiates two methodical categories for testing measurement accuracy of dispensers in its 4th draft from 1996. These are the gravimetric and non-gravimetric methods. Since not every laboratory has available sufficient balanced weighing stations and costly scales with the necessary resolution (six decimal places) for performing gravimetric measurements, photometric tests for hand pipettes, e.g. for the range of sample volumes from 0.2 to 1 μl, have been offered by the industry (e.g. the firm EPPENDORF AG, Barkhausenweg 1, D-22339 Hamburg, Germany).
A further method is known from the article “Performance Verification of Small Volume Mechanical Action Pipettes” by Richard H. Curtis [Cal.Lab, May/June 1996]. In consideration of the difficulties (e.g. evaporation, vibrations, static charge of the samples) of the application of gravimetric methods to a liquid sample in the microliter range, in this article an integrated system was suggested based on using calorimetric substances. However, the concentration of indicator substance whose optical density is to be measured must be known exactly. This optical density is calculated as log10 (1/T), with T referred to as transmittance. This transmittance corresponds to I/I0, i.e. the ratio of output intensity and input intensity of the light beams penetrating the sample. Furthermore, the device used for measuring the optical density must also meet international norms. In addition, problems such as a dependence of the measurement on the sample temperature, the appearance of changes in the solution, and the appearance of wear in the measurement cuvette must be considered. The firm ARTEL Inc., 25 Bradley Drive Westbrook, Me., USA, produces the “Artel PCS™ Pipette Calibration System” of this type. It essentially consists of a prefilled, sealed test glass with 4.75 ml of an exactly defined concentration of a copper chloride solution and an instrument for measuring the optical absorption (wherein optical absorption A=I0/I=−log10 T) of this solution at a wavelength of 730 nm. The test glass is inserted in the instrument and remains in place during the entire calibration process. The experimenter opens the seal of the test glass and adds a sample corresponding to the desired measurement precision to the glass with the pipette to be checked, and then reseals the seal. The sample added is a solution of “Ponceau S”, an organic test substance, which, among other things, is selected due to its long-term stability and good “pipettability” (similar to water, even at high concentrations) and its wide, well-defined absorption peak at 520 nm. The absorption peaks of the copper chloride solution and of the test solution “Ponceau S” do not overlap. In addition, the test solution contains biocides, in order to prevent the growth of microorganisms, and a pH-stabilizing buffer. The device mixes the two solutions with an integrated mixer and determines the absorption at 520 nm (Ponceau S) and at 730 nm (copper chloride). The volume of the sample added is then calculated on the basis of these two measured values and the known initial concentrations. Although this system has the advantage that the measurement of the optical absorption is allowed independently of the path length and irregularities in the test glass, it nonetheless has the disadvantage that it cannot be adapted at a reasonable expense for usage in a multichannel pipetting robot.
A further calibration method of this type uses “Orange G” as the test substance, which allows an absorption measurement with high sensitivity. However, it is disadvantageous in this case that the flat molecules of this test substance have a high adhesion to the inner walls of the pipette tip and/or to the tubings, troughs, and/or wells of microplates. Therefore, an uncontrollable reduction of the Orange G concentration in the test liquid occurs, which makes the reliability of the test questionable.
Another method of this type is known from Belgian patent No. BE 761 537, which describes the analysis of various substances with increased precision, particularly automatic analysis, which depends on the sample volume of the substance. According to this invention, one mixes chromium in the form of Cr2(SO4)3·10H2O into a sample as an indicator, in order to obtain a specific concentration of chromium (III) therein. With reference to the chromium (III) concentration measured, the effective volume of the sample is calculated. The sample volumes are in the milliliter range. Cr2(SO4)3·10H2O exists in aqueous solution as [Cr(H2O)6]3+. According to the literature (see, for example, W. Schneider in “Einführung in die Koordinationschemie”, Springer Verlag Berlin, Heidelberg, New York 1968, pp. 115-117), the aqueous complex [Cr(H2O)6]3+ has a molar extinction coefficient (ε) of only approximately 13 (where an ε of less than 100 is a low to average value). In pure aqueous complexes, the extinction coefficient ε is approximately 50. The concentration of a pigment and the optical absorption are linked via the Beer-Lambert law(A=c*ε*/).where:                A=optical absorption        c=concentration of the dissolved material [M=Mol/L]        ε=molar extinction coefficient of the dissolved material [1/(M·cm)]        l=layer thickness (the liquid which the light must pass through) [cm].Due to limitations in spectrophotometric hardware reasons, the art (cf. Bruno Lange et al. in “Photometrische Analyse”, VCH Verlagsgesellschaft mbH, Weinheim, 1987, p. 21) recommends that measurements only be performed in the absorption range from 0.1 to 1. The sensitivity of the measurement system increases with higher ε. In order to be able to measure a volume of 1 μl in a final volume of 200 μl with an optical absorption of 0.1, the concentration of [Cr(H2O)6]3+ must be at least 15 Mol/L according to the Beer-Lambert law. However, the physical properties of the sample are significantly changed by these high concentrations, and this, of course, is undesirable.        